The perlecan rich extracellular matrix (ECM) in bone marrow generally does not turn over rapidly except in the presence of inflammation or invasive cells. Invading metastatic cells can produce inflammation and so possess both of these features. Metastatic cell invasion of tissue releases enzymes that degrade extracellular matrix and allow expansion of tumor mass into surrounding tissue. Metastatic cancer cells produce heparanase, sulfatases, metalloproteinases (MMPs) such as 72- and 92-kD type IV collagenases (MMP-2, MMP-9 activated by bone), uPA, kallikrein 14, PSA, other MMPs such as stromelysins and matrilysins (MMP-3,7,10), and other collagenases such as MMP-1. BMP1/TLL are secreted MMPs, produced by alternative splicing of the TLL1 gene, that cleave fibrillar and non-fibrillar collagens, certain growth factors, α2-macroglobulin, lysyl oxidases, laminin, and several proteoglycans including perlecan. Gonzalez, et al., J. Biol. Chem. 280: 7080-7087, 2005.
Reactive oxygen species (ROS) increase production of and activate MMPs which degrade perlecan into smaller fragments with a wide range of potential bioactivities, and levels of ROS are higher in the vicinity of tumor tissue. Recently, Mauri, et al. have described proteins released by cancer cells that are involved in ECM remodeling, including HSPG2/perlecan, syndecan 4, and β2M. Mauri, et al., Faseb J. 19: 1125-1127, 2005.
Perlecan is a large, multifunctional, five domain heparan sulfate proteoglycan found in nearly all basal laminae as well as in the interstitial matrix of certain tissues, including bone marrow stroma. Farach-Carson et al., Crit. Rev. Eukaryot. Gene Expr. 15: 29-48, 2005; Noonan, et al., J. Biol. Chem. 266: 22939-22947, 1991. Perlecan is expressed constitutively at high levels in bone marrow and is the most abundant heparan sulfate proteoglycan (HSPG) in bone marrow extracellular matrix (Schofield, et al., Biochem J. 343: 663-668, 1999). As metastatic cancer cells invade, grow and reproduce in bone marrow, they release enzymes that degrade perlecan. This proteolytic degradation yields small perlecan fragments, which have ready access to the circulatory system from the bone marrow (Kopp, et al., Physiology 20: 349-356, 2005). In addition, degradation of perlecan generates bioactive fragments, such as endorepellin, with unique activities distinct from intact perlecan, including modulation of angiogenesis. Bix et al., J. Cell Biol. 166: 97-109, 2004.
The exact identities of most perlecan derived fragments have never been reported. Whitelock, et al., reported the degradation of perlecan by several proteases and glycosidases but did not identify the fragments. Whitelock, et al., J. Biol. Chem. 271: 10079-10086, 1996. Farach-Carson and Carson have described enzymes that may degrade perlecan, based on known enzyme target sequences in the perlecan protein. Glycobiology 9:897-905, 2007. These are shown in FIG. 1. The large number of proteolytic sites near the C-terminus of perlecan are actively cleaved by the same proteases that are produced by invading cancer cells at sites of tissue invasion, and perlecan expression increases in cancer tissues and cancer cell lines. Fjeldstad and Kolset, Current Drug Targets 6: 665-682, 2005; Lynch and Matrisian, Differentation 70: 561-573, 2002; Zhao, et al., J. Biol. Chem. 278: 15056-15064, 2003; Datta, et al., Molecular Cancer 5: 9-23, 2006. However, the sequences of most proteolytic sites in matrix proteins remain unknown, as are the exact identities of proteolytic fragments of perlecan. Additionally, because sequential cleavage by multiple proteases can occur, many novel perlecan-derived fragments can be created and enter the circulatory system.